Carbon dioxide is an end product of cellular metabolism. It is expired in humans at a rate of 9 mmol/kg-hour (1) The rate of 13CO2 production form 13C-labeled substrates has been demonstrated in cells, tissues, perfused organs and whole animals since the 1940s (2). Moreover, this approach has been used in biomedicine to measure liver function, malabsorption, bacterial infection, enzyme deficiency, pancreatic insufficiency and protein metabolism.
The principle of 13CO2 breath tests is to administer a substrate labeled with 13C either orally or intravenously. The substrate must possess a target bond that is attacked by a specific enzyme whose activity is to be measured. The enzymatic cleavage of the 13C bond is the rate limiting step. Ultimately, the 13C moiety is directly hydrolyzed or rapidly converted to 13CO2.
Existing CO2 tests generally require large amounts of labeled substrate. Tests based on radioactive labels are problematic because the patient consumes radioactive material. Disposal and handling costs also increase with radioactive labels. If non-radioactive labels are employed, the problems are not eliminated because labeled substrates are very expensive, thus increasing the costs of such tests significantly. What is needed in the art is a method that decreases the amount and cost of label required for a metabolic test, without sacrificing the needed sensitivity. The invention described herein, fulfills this need and, although exemplified with respect to a lactose intolerance assay, can be used wherever CO2 breath tests are used. The invention can also be used for metabolites other than CO2 and for samples other than breath samples.
Lactose maldigestion is the inability to digest significant amounts of lactose, the predominant sugar of milk. This inability results from a shortage of the enzyme lactase that is normally produced by the cells that line the small intestine. When there is not enough lactase to digest the amount of lactose consumed, the results may be very distressing and can result in dangerous dehydration among children. Common symptoms include nausea, cramps, bloating, gas, and diarrhea, which begin about 30 minutes to 2 hours after eating or drinking foods containing lactose. The severity of the symptoms varies depending on the amount of lactose each individual can tolerate.
The intestinal enzyme lactase (β-D-galactosidase) is responsible for metabolizing lactose. At birth, humans have abundant lactase activity in the small intestine but in most ethnic groups this activity decreases significantly during childhood between ages 3 to 5. Under conditions of lactase deficiency the lactose passes unmetabolized through the small intestine, drawing in copious amounts of water by osmosis. Next, the lactose passes into the large intestine and is fermented by colonic bacteria. Through these two processes, osmosis and fermentation, the typical symptoms associated with lactose maldigestion such as bloating, cramping, excessive gas and explosive diarrhea are derived.
Milk and other dairy products are a major source of nutrients in the American diet. The most important of these nutrients is calcium. Calcium is essential for the growth and repair of bones throughout life, but is a particular concern during the developmental years. In the middle and later years, a shortage of calcium may lead to thin, fragile bones that break easily; a condition known as osteoporosis. A concern, then, for both children and adults with lactose maldigestion, is getting enough calcium in a diet that contains little or no milk.
Studies have shown that nearly 50% of people who self-report milk intolerance are not maldigesters (1-3). Instead, they suffer from a functional bowel disorder such as irritable bowel syndrome (IBS), recurrent abdominal pain (RAP) in children or some other gastrointestinal complication. In these self-reported milk intolerants, it has been found that there is a significant, unnecessary reduction in milk consumption and insufficient dietary calcium intake (4).
Lactose maldigestion is relatively easy to treat. No treatment exists to improve the body's ability to produce lactase, but the symptoms can be controlled. Many foods are now available that are lactose-reduced or even lactose-free. Moreover, chewable tablets of lactase are available without prescription that, when taken just prior to a lactose-containing meal, can alleviate many symptoms.
However, all of these proposed therapies and remedies are only advisable in the person who is truly a lactose maldigesters (truly deficient in the enzyme lactase). For the person who suffers, for example, from irritable bowel syndrome (IBS) but is misdiagnosed as lactase-deficient, the addition of lactase in the form of tablets or the change to lactose-free dairy products will not alleviate symptoms. Moreover, those self-treaters who avoid dairy under the mistaken impression that they are maldigesters, put themselves at risk for poor bone growth and repair, osteoporosis and other conditions that results from the unnecessary removal of dairy products from their diet.
The diagnosis of lactose maldigestion has relied on the interview process coupled with removing milk (and milk products) from the diet, laboratory tests and jejunal biopsy. We briefly describe the state of each measure.
The interview process during which a patient is quizzed as to the history of their gastrointestinal symptoms and its relation to milk intake is easy to perform and inexpensive. It is also overly simplistic and quite imprecise. First, nearly 50% of people who self-report milk intolerance are normal digesters of lactose and secondly, 70% of the people with lactase-deficiency (although symptomatic) fail to correlate the broad gastrointestinal symptoms of this disease to the intake of lactose or “milk sugar” (7).
A number of laboratory tests are available for the assessment of lactose maldigestion. The most often cited tests are the hydrogen breath test, lactose tolerance test and the stool acidity test. The hydrogen breath test measures the amount of hydrogen in the breath. Normally, very little hydrogen is detectable in the breath. However, in the case of the lactose maldigesters, the lactose passes into the colon unmetabolized where bacteria ferment it and various gases, including hydrogen are produced. The hydrogen is absorbed from the intestines, carried through the blood stream to the lungs and exhaled. In this test, the patient drinks a lactose-loaded beverage, and the breath is analyzed at regular intervals over several hours. Raised levels of hydrogen in the breath indicates that the lactose is not being properly digested.
The interpretation of the hydrogen breath test results can be confounded by a number of factors. First, 5-20% of maldigesters do not produce hydrogen, resulting in a lowered sensitivity for the test (8). A comparable percentage of non-producers has been found in children (9). This is due to either not having the flora capable of producing hydrogen or utilization of the hydrogen to produce methane. Secondly, careful patient preparation, including no teeth brushing on the morning of the exam, no smoking, sleeping or strenuous activity during the exam is absolutely mandatory in order to produce a reliable test (10). Also, for one month prior to the test, there should be no mechanical bowel cleansing or antibiotic use since both influence the type and quantity of colonic bacteria (10). Finally, a low carbohydrate, low fiber dinner the night before the test is advised. Any deviations from these recommendations will compromise the test.
In the lactose tolerance test, a fasted individual (>10 hours without eating) is given a liquid that contains a large lactose load (typically 2g/kg to a maximum of 50 g which is equivalent to the lactose content of one liter of milk). Several blood samples are taken over a period of two hours to measure the subject's blood glucose level. This result is used as an indication of how well that patient digests lactose.
Again, there are several drawbacks to this test method. This test uses a supraphysiological dose of lactose, which makes its generalization to normal milk or dairy ingestion questionable. It requires a minimum of four (4) needle sticks over 2 hours to measure glucose concentration and strict patient compliance to a fasted state. Moreover, it suffers from decreased specificity (13% false positive rates have been reported) since a flattened response requires differentiation from defective glucose absorption resulting from small bowel disease (11). It has been suggested in the medical literature that due to both false negative and false positive results “that routine estimation of blood glucose after lactose load is not a useful measurement in children and adults and should be abandoned” (12).
In a recent study, it was shown that in 300 subjects tested using both the hydrogen breath test and the lactose tolerance test, in 40% of the cases, the two tests did not agree (13). The study suggests, however, that the hydrogen breath test is better able to identify individuals with lactose malabsorption and those most likely to have symptoms.
Due to the required lactose loads in these two diagnostic tests and the associated danger from dehydration resulting from lactose-induced diarrhea, they are generally not used in infants and very young children. Infants and young children may instead be given the stool acidity test. Undigested lactose, fermented by bacteria in the colon, creates lactic acid and other short-chain fatty acids that can be detected in a stool sample. This test is only effective in completely lactose-dominated diets (such as infant formula or breast milk) and since the incidence of lactose maldigestion in infants is very low, it is not often utilized.
Jejunal biopsy is an effective method for establishing a level of a patient's lactase activity. However, it is highly invasive and used only on rare occasion. Because lactose maldigestion is not generally considered a dangerous health condition, such an expensive, invasive and uncomfortable procedure is not a useful alternative.
Thus, what is needed in the art is a reliable, sensitive lactose intolerance test that is non-invasive, cost effective and accurate. The reverse isotope dilution test, exemplified herein with respect to lactose intolerance, meets these needs.